Shiga toxin (Stx) producing E. coli (STEC) are foodborne pathogens that can cause severe morbidity and mortality, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) and remain a major challenge for food safety and public health. There are no specific therapeutics or vaccines against infection by bacteria producing these toxins. Shiga toxins are a family of AB5 toxins consisting of an A1 subunit, which depurinates a universally conserved adenine in the ribosome, and a pentamer of identical B subunits, which bind to a glycolipid globotriaosylceramide receptor. E. coli strains producing Shiga toxin 2 (Stx2) are associated with progression to more severe disease than strains producing Shiga toxin 1 (Stx1). The mechanism that accounts for the differences in toxicity of Stx1 and Stx2 is not known. Although several studies suggested a role for the B subunit in the increased toxicity of Stx2, the role of the A1 subunit in the higher toxicity of Stx2 has not been investigated. The proposed studies seek to fill a critical gap in our understanding of Stx toxicity by examining the role of the A1 subunit. We propose that the A1 subunit in combination with the B subunit contributes to toxicity. We recently demonstrated that the A1 subunit of Stx2 has higher affinity for mammalian ribosomes and is more active than the A1 subunit of Stx1 and that differences in surface charge are critical for the interaction of the A1 subunits with ribosomes. We will test the hypothesis that ribosome binding kinetics and catalytic activity, which determine the rate of depurination, contribute to the differential toxicity of Stx1 and Stx2. In Aim 1 we will identify the residues that contribute to differences in surface charge of the A1 subunits and determine if they contribute to the differential activity and toxicity; in Aim 2, we will determine if depurination activity of the A1 subunit contributes to the lethality of Stx2 holotoxin in an animal model; and in Aim 3 we will identify peptides that can bind to ribosome docking surfaces and determine if they reduce toxicity of Stx2 by disrupting its interaction with the ribosome. This approach has already culminated in peptides that inhibit depurination activity of Stx2. We expect that our unique approach, the powerful set of tools we developed, ability to dissect the toxin activity in vitro and in vivo will reveal the factors critical for STEC pathogenesis and will provide a path to new therapy.